Methods of treating cancer of the central nervous system

ABSTRACT

A method of treating a cancer of the central nervous system in a subject in need thereof is provided. The method comprising administering to the subject a therapeutically effective amount of an agent which reduces blood glutamate levels and enhances brain to blood glutamate efflux to thereby treat the cancer of the central nervous system in the subject.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/685,628 filed on Apr. 14, 2015, which is a division of U.S. patentapplication Ser. No. 12/994,762 filed on Nov. 25, 2010, now U.S. Pat.No. 9,034,319, which is a national phase of PCT Patent Application No.PCT/IL2008/000711 having International Filing Date of May 26, 2008. Thecontents of the above Applications are incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates methods oftreating cancer of the central nervous system.

Central nervous system malignant neoplasias and glioma in particular,have so far defied all current therapeutic modalities. In the last fewyears, an ever increasing body of data have suggested that glutamate(Glu), the major excitatory neurotransmitter in brain, plays a crucialrole in the growth of malignant gliomas, their invasiveness and abilityto destroy neighboring brain tissue (Lyons, et al. 2007; Sontheimer2003) while being also the possible cause of the tumor-associatedseizures that often occur in conjunction with gliomas.

Thus, studies with glioma cells in culture have shown that the cellsrelease massive amounts of Glu resulting in elevations of theextracellular concentrations of Glu in excess of 100 μM within hours ina space that is 1000-fold larger than the cellular volume (Ye andSontheimer 1999). This massive release was attributed in part to adeficient Glu uptake by glioma cells attributed to thereduction-mislocalization of Glu transporters (Ye, et al. 1999).Moreover, exposure of cultured hippocampal neurons to glioma-conditionedmedium caused widespread neuronal death attributed to the excitotoxiceffects exerted by excess Glu (Ye and Sontheimer 1999). This neuronaldeath was inhibited by NMDA receptor antagonists as well as byinhibitors of the glutamate-cystine exchanger (Ye and Sontheimer 1999).The importance of this massive Glu release was explored in the contextof glioma growth. It was found that increased peritumoral levels of Gluwere present in the brain of rats implanted with RG2 glioma cells(Sheline, et al. 2000). Moreover, injection of C6 gliomas into rat brainstriata yielded significant tumor growth only in glioma cells capable ofreleasing Glu (Takano, et al. 2001).

Additional findings showed that antagonists of the NMDA and AMPAreceptors, two major subtypes of ionotropic Glu receptors, inhibited theproliferation (Rzeski, et al. 2002) and migration of both rat (Takano,et al. 2001) and human (Ishiuchi, et al. 2002) malignant gliomas whilean antagonist of the Glu metabotropic receptor reduced glioma growth(Arcella, et al. 2005).

Several scientific reports suggested glutamate inhibitors for thetreatment of gliomas include: Birmingham 2002; Ikonomidou and Turski2002; Lutsep and Clark 2001a; Lutsep and Clark 2001b. Related patentliterature include: U.S. Patent Application Number 20030050224.

PCT Publication Number WO2004/012762 teaches a method of reducingextracellular brain glutamate levels. The method comprises administeringto a subject in need thereof a therapeutically effective amount of anagent capable of reducing blood glutamate levels thereby reducingextracellular brain glutamate levels. Other related publicationsinclude, Pawlik, et al. 1981, O'Kane, et al. 1999, Gottlieb, et al.2003, (eichberg, et al. 2008 and Zlotnik, et al. 2008.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of treating a cancer of the central nervous system in a subjectin need thereof, the method comprising administering to the subject atherapeutically effective amount of an agent which reduces bloodglutamate levels and enhances brain to blood glutamate efflux to therebytreat the cancer of the central nervous system in the subject.

According to one aspect of the present invention there is provided a useof an agent which reduces blood glutamate levels and enhances brain toblood glutamate efflux for treating a cancer of the central nervoussystem.

According to one aspect of the present invention there is provided anarticle-of-manufacture comprising packaging material and apharmaceutical composition identified for treating a cancer of thecentral nervous system being contained within the packaging material,the pharmaceutical composition including, as an active ingredient, anagent capable of reducing blood glutamate levels and an anti canceragent and a pharmaceutically acceptable carrier.

According to further features in some embodiments of the inventiondescribed below, the anti cancer agent comprises a chemotherapy.

According to still further features in the described exemplaryembodiments the agent is at least one glutamate modifying enzyme and/ora modification thereof.

According to still further features in the described exemplaryembodiments the at least one glutamate modifying enzyme is selected fromthe group consisting of a transaminase, a dehydrogenase, adecarboxylase, a ligase, an aminomutase , a racemase and a transferase.

According to still further features in the described exemplaryembodiments the transaminase is selected from the group consisting ofglutamate oxaloacetate transaminase, glutamate pyruvate transaminase,acetylornithine transaminase, ornithine-oxo-acid transaminase,succinyldiaminopimelate transaminase, 4-aminobutyrate transaminase,(s)-3-amino-2-methylpropionate transaminase, 4-hydroxyglutamatetransaminase, diiodotyrosine transaminase, thyroid-hormone transaminase,tryptophan transaminase, diamine transaminase, cysteine transaminase,L-Lysine 6-transaminase, histidine transaminase, 2-aminoadipatetransaminase, glycine transaminase, branched- chain-amino-acidtransaminase, 5-aminovalerate transaminase, dihydroxyphenylalaninetransaminase, tyrosine transaminase, phosphoserine transaminase, taurinetransaminase, aromatic-amino-acid transaminase,aromatic-amino-acid-glyoxylate transaminase, leucine transaminase,2-aminohexanoate transaminase, ornithine(lysine) transaminase,kynurenine-oxoglutarate transaminase, D-4-hydroxyphenylglycinetransaminase, cysteine-conjugate transaminase, 2,5-diaminovaleratetransaminase, histidinol-phosphate transaminase,diaminobutyrate-2-oxoglutarate transaminase, andudp-2-acetamido-4-amino-2,4,6-trideoxyglucose transaminase.

According to still further features in the described exemplaryembodiments the dehydrogenase is a glutamate dehydrogenase.

According to still further features in the described exemplaryembodiments the decarboxylase is a glutamate decarboxylase.

According to still further features in the described exemplaryembodiments the ligase is a glutamate-ethylamine ligase.

According to still further features in the described exemplaryembodiments the transferase is selected from the group consisting ofglutamate N-acetyltransferase and adenylyltransferase.

According to still further features in the described exemplaryembodiments the aminomutase is a glutamate-1-semialdehyde2,1-aminomutase. According to still further features in the describedexemplary embodiments the agent is at least one co-factor of a glutamatemodifying enzyme.

According to still further features in the described exemplaryembodiments the co-factor is selected from the group consisting ofoxaloacetate, pyruvate, NAD⁺, NADP⁺, 2-oxohexanedioic acid,2-oxo-3-sulfopropionate, 2-oxo-3-sulfinopropionic acid,2-oxo-3-phenylpropionic acid, 3-indole-2-oxopropionic acid,3-(4-hydroxyphenyl)-2-oxopropionic acid, 4-methylsulfonyl-2-oxobutyricacid, 3-hydroxy-2-oxopropionic acid, 5-oxopentanoate, 6-oxo-hexanoate,glyoxalate, 4-oxobutanoate, α-ketoisocaproate, α-ketoisovalerate,α-keto-β-methylvalerate, succinic semialdehyde-(-4-oxobutyrate),pyridoxal phosphate, pyridoxal phosphate precursors and3-oxoisobutanoate.

According to still further features in the described exemplaryembodiments the agent comprises a glutamate modifying enzyme and aco-factor thereof.

According to still further features in the described exemplaryembodiments the glutamate modifying enzyme comprises glutamateoxaloacetate transaminase and the co-factor thereof comprisesoxaloacetate.

According to still further features in the described exemplaryembodiments the glutamate modifying enzyme comprises pyruvatetransaminase and the co-factor thereof comprises pyruvate.

According to still further features in the described exemplaryembodiments the administering is effected at a concentration of theagent not exceeding 1 g/Kg body weight/hour.

According to still further features in the described exemplaryembodiments the agent is at least one inhibitor of a glutamatesynthesizing enzyme.

According to still further features in the described exemplaryembodiments the inhibitor is selected from the group consisting ofgamma-Acetylenic GABA, GABAculine, L-canaline,2-amino-4-(aminooxy)-n-butanoic acid, 3-Chloro-4-aminobutanoate,3-Phenyl-4-aminobutanoate, Isonicotinichydrazide;(S)-3-Amino-2-methylpropanoate, Phenylhydrazine;4-Fluorophenyl)alanine, Adipate, Azaleic acid, Caproate,3-Methylglutarate, Dimethylglutarate, Diethylglutarate, Pimelate,2-Oxoglutamate, 3-Methyl-2-benzothiazolone hydrazone hydrochloride,Phenylpyruvate, 4-hydroxyphanylpyruvate, Prephenate and Indole pyruvate.

According to still further features in the described exemplaryembodiments the cancer is a glioma.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Copies of this patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a graph showing oxaloacetate effect on glioma cells in vitro.

FIGS. 2a-2b are magnetic resonance images of C6 glioma tumor cellsimplanted in the rat striatum. The T2 images illustrate serial coronalsections through the tumor. Parallel T1 images are shown on the leftpanel. As can be seen the tumor visualized on day 7 in a single brainsection (FIG. 2A) appeared 10 days later in 2 sections out of the foursequential sections presented (FIG. 2B, note that only the T2 images arepresented). The treatment started on day 7 comprised providing the ratswith either 0.2M oxaloacetate (in tap water) or 0.4M NaCl (in tap water)in their drinking water. In control animals which were treated withdrinking water alone, the tumor size was at least 10 times greater (notshown).

FIG. 3 is a graph showing glioma invasiveness in rats drinking 0.4 MNaCl (n=19, circular dots) and in rats drinking 0.2 M oxaloacetate(O×Ac, n=19, square dots). The tumor invasiveness was calculated bydividing the tumor size determined at the second MRI session by thatmeasured 10 days earlier at the first MRI session. (unpaired student ttest: p=0.02; F test to compare variances: p=0.001).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to methodsof treating cancer of the central nervous system.

For purposes of better understanding some embodiments of the present

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

While reducing the present invention to practice, the present inventoruncovered that a blood glutamate scavenger which enhances brain-to-bloodglutamate efflux can be used to inhibit the spread of tumor cells bypreserving normal brain cells which would otherwise be damaged or killedby tumor-derived glutamate.

The present inventors hypothesized that since brain tumors are highlyvascularized and brain capillary endothelial cells are involved in abrain-to-blood glutamate efflux, agents which are able to decrease bloodglutamate levels that in-turn increase the driving force for the effluxof excess glutamate from brain into blood should also cause theelimination of peritumoral excess glutamate. Accordingly it wassuggested that the elimination of peritumoral excess glutamate shouldimpede the ability of the tumor to create the necessary space for itsexpansion in brain.

As is illustrated hereinbelow and in the Examples section which follows,oral administration of oxaloacetate into rats implanted with gliomacells slowed tumor cell spread, as evidenced by MRI analysis.

Thus, according to one aspect of the present invention there is provideda method of treating a cancer of the central nervous system in a subjectin need thereof. The method comprising administering to the subject atherapeutically effective amount of an agent which reduces bloodglutamate levels and enhances brain to blood glutamate efflux and treatthe cancer of the central nervous system in the subject.

As used herein the phrase “cancer of the central nervous system” refersto a brain tumor (primary or secondary), which typically releasesglutamate at levels sufficient to allow glutamate to exertexcitotoxicity on neighboring healthy neuronal cells. Thus, a cancer ofthe central nervous system include, primary tumors of glial, neuronal,schwann cell, pinealcyte, menningioma and melanoma, as well as sarcoma,lymphoma and multiple systemic malignancies that metastasize in thebrain.

Specific examples include, but are not limited to, astrocytoma andglioblastoma, and also their related neural and glial tumors, glioma(e.g., which include grades 1 and 2), oligodendroglioma, neurocytoma,dysplastic neuroepithelial tumor, primitive neuroectodermal tumor, andganglioneuroma.

Other types of cancer which can be treated in accordance with thepresent teachings, can be identified using methods which are well knownin the art. For example, EAAT2 promoter activity assay, glutamatetoxicity assay (e.g., co-culturing with neurons followed by assayingcell killing), expression of glutamate cysteine exchanger glutamatetransporters (e.g., GCG, EAAC1, the latter as exemplified by U.S. PatentApplication Number 20030050224, which is hereby incorporated byreference). Alternatively, other cancers which can be subject to thepresent treating modalities can be those in which glutaminergic etiologyof intractable seizures and/or secondary brain atrophy, by induction ofapoptotic mechanisms is observed.

As used herein the term “treating” refers to abrogating, substantiallyinhibiting, slowing or reversing the progression of cancer of thecentral nervous system, substantially ameliorating clinical oraesthetical symptoms of a cancer of the central nervous system orsubstantially preventing the appearance of clinical or aestheticalsymptoms of a cancer of the central nervous system. In a specificembodiment the term treating refers to inhibiting or slowing spreading(i.e., growth) of a tumor of the central nervous system.

An agent, which is capable of reducing blood glutamate according to thisaspect of the present invention includes any glutamate modifying enzymeand/or a co-factor thereof or any artificially modified derivatives(e.g. esters).

Enzymes that use glutamate as a substrate follow the general formula:

A+GLUTAMATE 4←(enzyme)→C+D whereby A represents theco-substrate,)enzyme) symbolizes a reversible enzyme and C and D aremetabolites of the enzyme. Examples illustrated by this formula include:Glutamate+oxaloacetate ←(GOT)→2-keto-glutarate+aspartate,Glutamate+pyruvate 4←(GPT)→2-keto-glutarate+alanine orGlutamate+4-methyl-2-oxopentoate 4←(branched-chain-amino-acidtransaminase) 2-ketoglutarate+Valine.

Examples for different substrates that work on the same enzyme include:Glutamate+2-oxohexanedioic acid←(GOT)→2-keto-glutarate+2-aminohexanedioic acid.Glutamate+2-oxo-3-phenylpropionic acid←(GOT)→2-keto-glutarate+phenylalanine.Glutamate+3-hydroxy-2-oxopropionic acid ←(GOT)→2-keto-glutarate+serine.Glutamate+5-oxopentanoate 4←(GPT)→2-keto-glutarate+5-aminopentanoate.Glutamate+4-oxobutanoate 4←(GPT)→2-keto-glutarate+4-aminobutanoate.Glutamate+glyoxalate 4←(GPT)→2-keto-glutarate+glycine.

The conversion of glutamate to 2-ketoglutarate is reversible. Thus, uponglutamate transformation via an enzymatic reaction into 2-ketoglutarate,there is a buildup of 2-ketoglutarate which can cause the enzyme to workin the reverse direction and convert 2-ketoglutarate into glutamate. Itis therefore beneficial to further break down 2-ketoglutarate and inthis way ensure the continual metabolism of glutamate. One such enzymethat metabolizes 2-ketoglutarate is 2-ketoglutarate dehydrogenasethrough the general reaction—2-ketoglutarate+lipoamide ←(2-ketoglutaratedehydrogenase) S-succinyldihydrolipoamide+CO₂.

Thus, as used herein “a glutamate modifying enzyme” is an enzyme, whichutilizes glutamate as a substrate and produces a glutamate reactionproduct. A glutamate modifying enzyme can be a natural occurring enzymeor an enzyme which has been modified to obtain improved features, suchas higher affinity to glutamate than to a modified glutamate, stabilityunder physiological conditions, solubility, enhanced enantioselectivity,increased thermostability and the like as is further describedhereinunder.

Numerous glutamate modifying enzymes are known in the art. For example,transaminases, which play a central role in amino acid metabolism andgenerally funnel α-amino groups from a variety of amino acids via thecoupled conversion of glutamate into α-ketoglutarate or ofα-ketoglutarate into glutamate.

Examples of transaminases include but are not limited to glutamateoxaloacetate transaminases, glutamate pyruvate transaminases,acetylornithine transaminases, ornithine-oxo-acid transaminases,succinyldiaminopimelate transaminases, 4-aminobutyrate transaminases,alanine transaminases (note: same as glutamate pyruvate transaminases,(s)-3-amino-2-methylpropionate transaminases, 4-hydroxyglutamatetransaminases, diiodotyrosine transaminases, thyroid-hormonetransaminases, tryptophan transaminases, diamine transaminases, cysteinetransaminases, L-Lysine 6-transaminases, histidine transaminases,2-aminoadipate transaminases, glycine transaminases,branched-chain-amino-acid transaminases, 5-aminovalerate transaminases,dihydroxyphenylalanine transaminases, tyrosine transaminases,phosphoserine transaminases, taurine transaminases, aromatic-amino-acidtransaminases, aromatic-amino-acid-glyoxylate transaminases, leucinetransaminases, 2-aminohexanoate transaminases, ornithine (lysine)transaminases, kynurenine-oxoglutarate transaminases,D-4-hydroxyphenylglycine transaminases, cysteine-conjugatetransaminases, 2,5-diaminovalerate transaminases, histidinol-phosphatetransaminases, diaminobutyrate-2-oxoglutarate transaminases,UDP-2-acetamido-4-amino-2,4,6-trideoxyglucose transaminases andaspartate transaminases.

Other examples of glutamate modifying enzymes include but are notlimited to glutamate dehydrogenases, which generate ammonium ion fromglutamate by oxidative deamination; decarboxylases such as glutamatedecarboxylase; ligases such as glutamate-ethylamine ligase,glutamate-cysteine ligase; transferases such as glutamateN-acetyltransferase and N2-acetyl-L-ornithine, adenylyltransferase;aminomutases such as glutamate-1-semialdehyde 2,1-aminomutase andglutamate racemase [Glavas and Tanner (2001) Biochemistry40(21):6199-204)].

It will be appreciated that artificially modified enzymes can also beused according to this aspect of the present invention.

Modification of enzymes can be effected using numerous protein directedevolution technologies known in the art [for review see Kuchner andArnold (1997) TIBTECH 15:523-530; Furukawa J Bacteriol. 2004 August;186(16):5189-96].

Typically, directed enzyme evolution begins with the creation of alibrary of mutated genes. Gene products that show improvement withrespect to the desired property or set of properties are identified byselection or screening, and the gene(s) encoding those enzymes aresubjected to further cycles of mutation and screening in-order toaccumulate beneficial mutations. This evolution can involve few or manygenerations, depending on the progress observed in each generation.

Preferably, for successful directed evolution a number of requirementsare met; the functional expression of the enzyme in a suitable microbialhost; the availability of a screen (or selection) sensitive to thedesired properties; and the identification of a workable evolutionstrategy.

Examples of mutagenesis methods which can be used in enzyme directedevolution according to this aspect of the present invention include butare not limited to UV irradiation, chemical mutagenesis, poisonednucleotides, mutator strains [Liao (1986) Proc. Natl. Acad. Sci. U.S.A83:576-80], error prone PCR [Chen (1993) Proc. Natl. Acad. Sci. U.S.A90:5618-5622], DNA shuffling [Stemmer (1994) Nature 370:389-91],cassette [Strausberg (1995) Biotechnology 13:669-73], and a combinationthereof [Moore (1996) Nat. Biotechnol. 14:458-467; Moore (1997) J. Mol.Biol. 272:336-347].

Screening and selection methods are well known in the art [for reviewsee Zhao and Arnold (1997) Curr. Opin. Struct. Biol. 7:480-485; Hilvertand Kast (1997) Curr. Opin. Struct. Biol. 7:470-479]. Typically,selections are attractive for searching larger libraries of variants,but are difficult to device for enzymes that are not critical to thesurvival of the host organism. Further more, organisms may evade imposedselective pressure by unexpected mechanisms. Less stringent functionalcomplementation can be useful in identifying variants which retainbiological activity in libraries generated using relatively highmutagenic rates [Suzuki (1996) Mol. Diversity 2:111-118; Shafikhani(1997) Biol. Techniques 23:304-310; Zhao and Arnold (1997) Curr. Opin.Struct. Biol. 7:480-485].

As described hereinabove, the agent according to this aspect of thepresent invention, can include one or more co-factors of glutamatemodifying enzymes, which can accelerate activity of the latter(V_(max)). These can be administered in order to enhance the rate ofendogenous glutamate modifying enzymes or in conjunction with glutamatemodifying enzymes (described hereinabove).

Co-factors of glutamate-modifying enzymes include but are not limited tooxaloacetate, pyruvate, NAD⁺, NADD⁺, 2-oxohexanedioic acid,2-oxo-3-sulfopropionate, 2-oxo-3-sulfinopropionic acid,2-oxo-3-phenylpropionic acid, 3-indole-2-oxopropionic acid,3-(4-hydroxyphenyl)-2-oxopropionic acid, 4-methylsulfonyl-2-oxobutyricacid, 3-hydroxy-2-oxopropionic acid, 5-oxopentanoate, 6-oxo-hexanoate,glyoxalate, 4-oxobutanoate, α-ketoisocaproate, α-ketoisovalerate,α-keto-β-methylvalerate, succinic semialdehyde-(-4-oxobutyrate),3-oxoisobutanoate, pyridoxal phosphate, 5-oxopentanoate, 6-oxohexanoateand their artificially modified derivatives (e.g., esters).

Since modified glutamate (i.e., glutamate reaction product) can bereversibly modified (i.e., interconverted) to glutamate, the agent,according to this aspect of the present invention, preferably includes amodified glutamate converting enzyme which is incapable of convertingthe modified glutamate back into glutamate to thereby insuring continualmetabolism of glutamate.

Examples of modified or modifiable glutamate converting enzymes includebut are not limited to GPT, GOT, Glutamate decarboxylase and glutamatedehydrogenase.

Modified glutamate converting enzymes can also include glutamatemodifying enzymes artificially modified to possess lower affinity forglutamate reaction product than for glutamate. For example, the E. coliGOT (GenBank Accession No. D90731.1) is characterized by an affinity forglutamate of about 8 mM and an affinity for 2-ketoglutarate of about 0.2mM. A human enzyme or a humanized enzyme characterized by suchaffinities is preferably used according to this aspect of the presentinvention such as described by Doyle et al. in Biochem J. 1990270(3):651-7.

Optionally, co-factors of modified glutamate converting enzymes can beincluded in the agent according to this aspect of the present invention.Examples of co-factors of modified glutamate converting enzymes includebut are not limited to lipoic acid and its precursors, thiaminepyrophosphate and its precursors, pyridoxal phosphate and its precursorsand the like.

It will be appreciated that the agent according to this aspect of thepresent invention may also include inhibitors of glutamate synthesizingenzymes (e.g., phosphate activated glutaminase). Numerous inhibitors ofglutamate producing enzymes are known in the art. Examples include butare not limited gabapentin which has been shown to modulate the activityof branched chain aminotransferases [Taylor (1997) Rev. Neurol.153(1):S39-45] and aspirin at high doses (i.e., 4-6 g/day) aneuroprotective drug against glutamate excitotoxicity [Gomes (1998) Med.J. India 11:14-17]. Other inhibitors may be identified in the publiclyavailable BRENDA, a comprehensive enzyme information system[www(dot)Brenda(dot)uni-koeln(dot)de/]. Examples include but are notlimited to, gamma-Acetylenic GABA, GABAculine, L-canaline,2-amino-4-(aminooxy)-n-butanoic acid;;3-Chloro-4-aminobutanoate;3-Phenyl-4-aminobutanoate; Isonicotinic hydrazide;(S)-3-Amino-2-methylpropanoate; Phenylhydrazine; 4-Fluorophenyl)alanine;Adipate, Azelaic acid, Caproate, 3-Methylglutarate, Dimethylglutarate,Diethylglutarate, Pimelate, 2-Oxoglutamate; 3-Methyl-2-benzothiazolonehydrazone hydrochloride; Phenylpyruvate, 4-Hydroxyphenylpyruvate,Prephenate, Indole pyruvate and their artificially modified derivatives(e.g., esters). Although each of the components described hereinabovemay comprise the agent of the present invention, it will be appreciatedthat for optimal blood-glutamate reducing activity, the agent mayinclude a combination of the above described components (i.e., glutamatemodified enzyme, co-factor thereof, modified glutamate converting enzymeand co-factor thereof).

For example, co-factor molecules e.g., oxaloacetate, reaching the bloodsystem may be absorbed into tissues via the ubiquitously distributedtransporters (e.g., dicarboxylate transporters). Thus, in the case ofoxaloacetate, the blood glutamate scavenging ability of oxaloacetaterelies mainly on the levels of the glutamate-oxaloacetate transaminase(GOT) present in blood. Increasing the blood GOT concentration by thesystemic (e.g., intravenous) administration of recombinant GOT can thusbe expected to significantly improve the blood glutamate scavengingproperty of oxaloacetate. The latter effect should also benefit from amore effective administration of oxaloacetate in order to reach higherand steady levels of blood plasma oxaloacetate.

Thus, according to exemplary embodiments of this aspect of the presentinvention the agent includes oxaloacetate and pyruvate. Preferably, theagent is administered at a dose not exceeding 1 g/kg×hour.

In some cases, the agent administered is modified in order to increasethe therapeutic effect or reduce unwanted side effects. For example,administration of oxaloacetate diethylester is favorable overadministration of oxaloacetate alone since oxaloacetate exerts itstherapeutic potential at relatively high concentrations and requiresfull titration of its carboxyl moieties with sodium hydroxide at 3:1stoichiometric ratio which presents unacceptable electrolyte load abovesafe levels.

Alternatively or additionally, to increase the life-time of the enzymein the blood, the present invention also envisages repetitiveadministration of the enzyme (e.g., on a monthly basis), use of reagentswhich will increase the life-time in the circulation e.g,. PEGylatedenzyme, or the entrapment of the enzyme in suitable carriers that shouldcause a sustained increase of blood enzyme levels allowing an effectiveblood glutamate scavenging with lower concentrations of the co-factor.

Alternatively, recombinant or purified enzymes of the present inventionmay be attached to a heterologous polypeptide that increases thehalf-life of the enzyme in the serum. Examples of heterologous aminoacid sequences that may be used in accordance with the teachings of thepresent invention include, but are not limited to, immunoglobulin,galactosidase, glucuronidase, glutathione-S-transferase (GST), carboxyterminal peptide (CTP) from chorionic gonadotrophin (CG□) andchloramphenicol acetyltransferase (CAT) [see for example U.S.Publication No. 20030171551].

The agent can be administered to a subject using any one of severalsuitable administration modes which are further described hereinbelowwith respect to the pharmaceutical compositions of the presentinvention.

The agent utilized by the method of the present invention can beadministered to an individual subject per se, or as part of apharmaceutical composition where it is mixed with a pharmaceuticallyacceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described hereinabove along withother components such as physiologically suitable carriers andexcipients, penetrants etc. The purpose of a pharmaceutical compositionis to facilitate administration of a compound to an organism.

Herein the term “active ingredient” refers to the preparationaccountable for the biological effect (e.g., the glutamate modifyingenzyme, and/or cofactors thereof).

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” are interchangeably used refer toa carrier or a diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound. An adjuvant is included under these phrases.One of the ingredients included in the pharmaceutically acceptablecarrier can be for example polyethylene glycol (PEG), a biocompatiblepolymer with a wide range of solubility in both organic and aqueousmedia (Mutter et al. (1979).

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration of the pharmaceutical composition ofthe present invention may, for example, include oral, rectal,transmucosal, especially transnasal, intestinal or parenteral delivery,including intramuscular, subcutaneous and intramedullary injections aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, intraosseus and intraocular injections.

Preferred is a systemic mode and dosing that reduces blood (plasma)glutamate levels and enhances brain-to-blood glutamate levels.

The administration mode may also depend on the status of the patient.For example, in case of a seizure, intraosseus administration orintravenal administration may be preferred.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, for oralingestion by a patient. Pharmacological preparations for oral use can bemade using a solid excipient, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art.

For any pharmaceutical composition used by the treatment method of theinvention, the therapeutically effective amount or dose can be estimatedinitially from in vitro assays. For example, a dose can be formulated inanimal models and such information can be used to more accuratelydetermine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Animal models for cancer of the nervous system are readily available.

Thus, xenograft models are well suited for evaluating dose responsecharacteristics of preclinical therapies, and for assessing theinfluence of tumor site on therapeutic response.

Weissenberger et al J Neurosurg. 2007 April; 106(4):652-9 have producedgermline insertion of a transgene expressing v-src from the GFAPpromoter (limiting the expression to astrocytes) resulting in theformation of astrocytomas. V-src activates several signal transductionpathways that are also activated in human gliomas. These GFAP/v-srcgliomas are primarily either low grade or anaplastic but in some casesacquire the histologic characteristics of glioblastomas.

Guha et al. Am J Pathol. 2005 September; 167(3):859-67 have developedtransgenic mice which over-expressed oncogenic H-Ras from the GFAPpromoter as. Different founder lines of these mice express variouslevels of oncogenic Ras. The highest producers of H-Ras developastrocytomas with all the characteristics of glioblastomas. The moderateH-Ras expressors go on to germline transmission and develop low gradeand anaplastic astrocytomas with high prevalence by 3 months of age.

Reilly et al. Nat Genet. 2000 September; 26(1):109-13 have generatedgliomas with astrocytic character with a combined deletion of Nf-1 andp53. Nf-1 is a RasGAP protein that down regulates Ras activitytherefore, loss of Nf-1 results in elevated Ras activity. Alone,mutation of Nf-1 in all cells within the mouse results in astrogliosis,but not glioma formation; however, when combined with mutations of p53,mice develop astrocytic tumors with characteristics of glioblastomas inhumans.

Retroviral vector gene transfer of PDGF-B to somatic cells has been usedto generate astrocytic gliomas by Uhrbom et al. Nat Med. 2004 November;10(11):1257-60 In these experiments, replication-competent MMLV vectorsystems result in the formation of various CNS tumor morphologies. Themost frequent histology seen in these experiments are high grade gliomaswith characteristics of glioblastomas.

Somatic-cell gene transfer with tissue-specific ALV based RCASretroviral vectors also show the formation of glioblastomas in mice.These tumors arise after combined gene transfer of genes encodingactivated Ras and Akt to nestin expressing CNS progenitors. In thissystem, neither Ras nor Akt alone are sufficient for the generation ofthese glioblastomas.

A transgenic mouse model with features of human WHO grade IIIastrocytoma was developed by astrocyte-specific inactivation of pRb andrelated proteins, p107 and p130 (Xiao et al., 2002). This wasaccomplished by expression of a single copy of the T121 gene driven bythe GFAP promoter. T121 is a 121-amino acid N-terminal fragment of SV40T antigen that dominantly inactivates the pRb proteins, but does notinterfere with p53 function. One hundred percent of TgG(Z) T121 micedevelop high grade astrocytoma at around 6 months of age. Histologicfeatures resembling the human disease include adhesion to neurons andvasculature.

The Examples section, which follows provides further guidance as tosuitable dosages.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state or symptoms isachieved.

The amount of the pharmaceutical composition to be administered will, ofcourse, be dependent on the subject being treated, the severity of theaffliction, the manner of administration, the judgment of theprescribing physician, etc. Compositions including the preparation ofthe present invention formulated in a compatible pharmaceutical carriermay also be prepared, placed in an appropriate container, and labeledfor treatment of an indicated condition.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert.

It will be appreciated that the present agents can be provided (asadjuvant therapy) along with other treatment modalities for braintumors, which are selected based on location, the cell type and thegrade of malignancy. Conventional therapies include surgery, radiationtherapy, and chemotherapy. Temozolomide is a chemotherapeutic drug thatis able to cross the blood-brain barrier effectively and is being usedin therapy. For recurrent high-grade glioblastoma, recent studies havetaken advantage of angiogenic blockers such as bevacizumab incombination with conventional chemotherapy, with encouraging results.Other agents include, but are not limited to, temodal, nitrosoureas,carmustine and cis-platin as well as antibody-based drugs, e.g.,cetuximab.

Other Anti-cancer drugs that can be co-administered with the agents ofthe invention include, but are not limited to Acivicin; Aclarubicin;Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin;Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide;Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; BleomycinSulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin;Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; CarubicinHydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflornithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide;Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine;Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil;Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; GemcitabineHydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b;Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran;Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Sulofenur; Talisomycin; Taxol; Tecogalan Sodium; Tegafur; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiamiprine; Thioguanine; Thiotepa; Tiazofuirin; Tirapazamine; TopotecanHydrochloride; Toremifene Citrate; Trestolone Acetate; TriciribinePhosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin;Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide;Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine;Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride. Additional antineoplastic agents include those disclosedin Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A.Chabner), and the introduction thereto, 1202-1263, of Goodman andGilman's “The Pharmacological Basis of Therapeutics”, Eighth Edition,1990, McGraw-Hill, Inc. (Health Professions Division).

It is expected that during the life of a patent maturing from thisapplication many relevant enzyme and co-factor agents will be developedand the scope of the agents for lowering blood glu level is intended toinclude all such new technologies a priori.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”. This termencompasses the terms “consisting of” and “consisting essentially of”.

The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the claimed composition or method.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate some embodiments of the invention in anon limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Maryland (1989); Perbal, “A Practical Guideto Molecular Cloning”, John Wiley & Sons, New York (1988); Watson etal., “Recombinant DNA”, Scientific American Books, New York; Birren etal. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishelland Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H.Freeman and Co., New York (1980); available immunoassays are extensivelydescribed in the patent and scientific literature, see, for example,U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987;3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader.

Materials and Experimental Procedures

Animals—Male SPD&OLD rats weighing 260-280 g were used and placed inindividual cages. All experiments were conducted according to theguidelines of the institutional animal care and use committee of theWeizmann Institute of Science, Rehovot, Israel.

Cell cultures—The rat glioma cell line C6 (ATCC Accession NumberCCL-107) was cultured (10 ml in a T-25 cm2 flask) in DMEM supplementedwith 10% FBS, 0.1 mg/ml kanamycin, and 4 mg/mlglucose at 37° C. under ahumidified atmosphere of 5% CO2, 95% air. Glioma cells were subculturedevery 3-4 days. For each experiment, confluent monolayers of cells werereleased from the tissue culture flask using 0.25% trypsin and rinsedtwice with serum-free RPMI-1640. Cells were cultured in 96-well plates(20×10³/0.2 ml/well) or in 12-well plates (160×10³/3 ml/well) for thedetermination of cell viability and proliferation. Following anovernight adherence period, the 96-well cultured cells were rinsed twicewith serum-free RPMI-1640 and then treated with either vehicle oroxaloacetate for 24-72 h (4 wells/treatment group). Cell number wasdetermined using hemocytometer.

Intrastriatal implantation Rat C6 glioma cells were grown in DMEM mediumsupplemented with 10% FCS. 10⁷ cells were plated on 100×20 mm dishes andgrown to 80% confluence for two days, passaged at a dilution of 1:10after 1-2 min exposure to 0.25% trypsin and plated on 7 plates 60×15 mm.The cells were grown to 80% confluence for two additional days. Thecells were then trypsynized as above, washed once with sterile PBS andcentrifuged for 15 min at 1200 rpm. Prior to centrifugation, the cellswere counted in a hemocytometer. Following centrifugation, the mediumwas removed and the cells were diluted in sterile PBS to a finalconcentration 1×10⁴/ml and 1 ml was injected immediately intracranially.For that purpose, the rats were anesthetized with 1.5% Halothane andplaced in a stereotactic apparatus.

The glioma cells were slowly injected over 20 min into a 0.5 mm pocketmade with a 23-gauge needle in the left anterior corpus striatum (0 mmbregma, +3 mm left lateral, 5.5 mm depth). In some cases, rats receivedalso a subcutaneous injection. In that case, 1×10⁶ glioma cells wereinjected in the chest area.

Treatment protocol—The treatment protocol comprised providing the rats(each rat was placed into an individual cage) with drinks consisting of0.2M oxaloacetate or NaCl; The control group consisted of rats offered50 ml/day of 0.4M NaCl in tap water. The experimental group consisted ofrats offered 50 ml/day of 0.2M oxaloacetate sodium salt in tap water.The reason for the apparent difference in concentration is due to thefact that the solution of sodium oxaloacetate is extremely acidic andhas to be neutralized by an equivalent amount of NaOH in order to reachneutral pH values.

MRI analysis—Experiments on live animals were conducted on a BiospecAvance™ system (Bruker Biospin, Ettlingen, Germany) with a 4.7 T 30-cmhorizontal bore magnet. For the experiments described here, the mainactively shielded 20-cm i.d. gradient coil was used with a maximalintensity of 100 mT/m and 0.33-ms rise time. The RF probe systemincluded a 7.5-cm resonator for pulse transmission and a 2.5-cm circularreceive-only surface coil with active detuning between the transmit andreceive coils. MRI analysis of the growth of a C6 glioma implanted inrat brain striatum was performed using a Sequential T2-W andgadolinium-enhanced T1-W images in the tumor region. For the T1-Wimages, 0.5 ml of Dotarem (gadoteric acid 27.932 g, DOTA 20.246 g,gadolinium oxide 9.062 g per 100 ml of solution) was injectedintravenously.

The parameters for image acquisition were: time of repetition=2000 ms,echo time=54 ms, field of view=4.0 cm, matrix size=256×256, slicethickness 1.0 mm, slice gap between center to-center of consecutiveslices=1.2 mm. The volume of the tumor was assessed using the Surdriverprogram.

Results

Oxaloacetate does not affect glioma cells in vitro—To test the effect ofoxaloacetate (OA) on glioma cells in vitro, C6 glioma cells wereincubated with 1 mM oxaloacetate. Since oxaloacetate solutions areunstable, the cell culture medium was changed every day to allow thecells to be daily exposed to a fresh oxaloacetate solution. As can beseen from FIG. 1, viability and proliferative status of the glioma cellswas not affected by OA.

Oxaloacetate retards glioma spread in vivo—After establishing thatoxaloacetate has no effect on glioma growth when grown under in vitroconditions, the effect of same in vivo was determined. For that purpose,C6 glioma cells were implanted into the rat striatum. Tumor developmentwas followed using MRI T1 and T2 imaging. FIGS. 2a-b show an example oftumor growth in an oxaloacetate-treated rat. Tumor spread was in averageat least 10 folds less than that of untreated animals that receiveddrinking water only (not shown). The results on two groups of 19 ratsare shown in FIG. 3. Clearly, OA retards tumor invasivenss in asignificant manner.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

REFERENCES Other References are Cited Throughout the Application

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What is claimed is:
 1. A method of treating a cancer of the centralnervous system in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of aglutamate modifying enzyme and optionally a co-factor thereof whichreduces blood glutamate levels to thereby treat the cancer of thecentral nervous system in the subject.
 2. An article-of-manufacturecomprising packaging material and a pharmaceutical compositionidentified for treating a cancer of the central nervous system beingcontained within the packaging material, the pharmaceutical compositionincluding, as an active ingredient, an agent capable of reducing bloodglutamate levels and an anti cancer agent and a pharmaceuticallyacceptable carrier.
 3. The article of manufacture of claim 2, whereinthe anti cancer agent comprises a chemotherapy.
 4. The method of claim1, wherein the glutamate modifying enzyme comprises glutamateoxaloacetate transaminase and the co-factor thereof comprisesoxaloacetate.
 5. The method of claim 1, wherein the glutamate modifyingenzyme comprises pyruvate transaminase and the co-factor thereofcomprises pyruvate.
 6. The method of claim 1, wherein the cancer is aglioma.